Functional expression of a P2T ADP receptor in Xenopus oocytes injected with megakaryocyte (CMK 11-5) RNA.
Functional expression of a P2T ADP receptor in Xenopus oocytes injected with megakaryocyte (CMK 11-5) RNA.
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注射巨核细胞 (CMK 11-5) RNA 的爪蟾卵母细胞中 P2T ADP 受体的功能表达。
DOI：
10.1161/01.atv.17.4.769
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Greco,NJ
中科院分区：
文献类型：
--
作者：
Greco,NJ
由于P2 T嘌呤能（ADP）受体是巨核细胞/血小板谱系所特有的，因此筛选了该谱系的细胞，以确定ADP和ATP对细胞内Ca 2+水平的相对影响。与血小板一样，CMK 11-5细胞对ADP的反应是细胞内Ca 2+动员增加，但对ATP或腺苷没有反应。与此相反，这两种核苷酸增加巨核细胞系MO 7 E和Meg-01的细胞内Ca 2+动员，表明它们含有P2 Y受体或嘌呤能受体的混合补体。CMK 11-5细胞对核苷酸的药理反应性与血小板相似，其中ADP和ADP-α-S作为激动剂有活性，ATP和ATP-α-S作为拮抗剂无活性。[~ 3 H]ADP和~（35）S-ATP-α-S与CMK 11-5细胞的结合位点分别为高亲和力位点（Kd 1和Ki 1，分别为262和125 nmol/L）和低亲和力位点（Kd 2和Ki 2，分别为10 100和5400 nmol/L），每个细胞有2× 106 ~ 6× 106个位点。ADP与ADP、ATP和ATP-α-S竞争结合，其亲和力顺序与血小板相似（ATP-α-S → ATP → ADP≥ADP-β-S →腺苷），提示CMK 11-5细胞上存在P2 T受体。用~（35）S-ATP-α-S光亲合标记完整的CMK 11-5细胞，结果显示GP Ⅱ b的α亚基被标记，与血小板一样，尽管这被证实不依赖于ADP受体。将CMK 11-5细胞的RNA显微注射到异种卵母细胞后，只有ADP和ADP-α-S刺激45 Ca ~（2+）外排，而ATP、2-甲硫基-ATP、α，β-亚甲基-ATP、ATP-γ-S、ATP-α-S和腺苷均不能刺激45 Ca ~（2+）外排。此外，注射RNA的卵母细胞与ATP或ATP-α-S孵育，但不与腺苷孵育，阻断了ADP对45 Ca ~（2+）的反应。这些实验表明，一个新生的受体，特异性地响应ADP，但不响应其他P1，P2 Y，P2 X，和P2 U激动剂表达的功能形式在异种卵母细胞。
Since the P2Tpurinergic (ADP) receptor is unique to the megakaryocytic/platelet lineage, cells of this lineage were screened for the relative effects of ADP and ATP in intracellular Ca2+levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca2+mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca2+mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P2Yreceptors or a mixed complement of purinergic receptors. Pharmacological responsiveness of CMK 11-5 cells to nucleotides paralleled those of platelets, in which ADP and ADP-α-S are active as agonists and ATP and ATP-α-S are inactive as agonists but act as antagonists. [3H]ADP and35S-ATP-α-S bound to CMK 11-5 cells at a high-affinity site (Kd1andKi1, 262 and 125 nmol/L, respectively) and a low-affinity site (Kd2andKi2, 10 100 and 5400 nmol/L, respectively) with 2×106to 6×106sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP-α-S with affinities in a rank order similar to that found for platelets (ATP-α-S≈ATP≈ADP≥ADP-β-S≈adenosine), suggesting the presence of a P2Treceptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with35S-ATP-α-S resulted in the labeling of the α-subunit of GP IIb as found with platelets, although this was confirmed to be independent of ADP receptors. After RNA from CMK 11-5 cells was microinjected intoXenopusoocytes, only ADP and ADP-α-S stimulated45Ca2+efflux, which was not observed with ATP, 2-methylthio-ATP, α,β-methylene-ATP, ATP-γ-S, ATP-α-S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP-α-S but not adenosine blocked the45Ca2+response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P1, P2Y, P2X, and P2Uagonists was expressed in functional form onXenopusoocytes.
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